Saccharides are nature's conveyors of energy, and are essential for cell survival. James, T. D. et al., J. Am. Chem. Soc., 117, 8982-8987 (1995) (citing Robertson, R. N., The Lively Membranes, Cambridge University Press, New York, 1983). Defects in glucose transport are associated with various disease states, and fluctuations in glucose levels can be symptomatic or predictive of acute, as well as chronic conditions. Id. Thus, the ability to detect and monitor glucose levels in animals, particularly humans, is extremely important.
Molecular recognition of saccharides or sugars, such as glucose, has proven to be a reliable detection mechanism. Perhaps the most reliable and efficient systems for such detection are those exploiting the phenomena of intramolecular electron transfer, also called photoinduced electron transfer (PET).
Systems capable of effecting such molecular recognition exhibit both structural and fluorescence changes upon saccharide binding. The change in fluorescence is proportional to the concentration of saccharide in the sample, and so these sensors or dyes can be used for quantitative measurement.
Among the more popular of these sensors are boronic acid based saccharide sensors. Boronic acid sensors effect reversible formation of strong covalent bonds with the diol functionalities of carbohydrates in the form of cyclic esters. These sensors are superior to other sensor systems involving weaker noncovalent or hydrogen bonded interactions.
Boronic acid sensors are not without their own shortcomings. The design of a fluorescent sensor based on the boronic acid-saccharide interaction proved difficult due to the lack of sufficient electronic changes found in either the boronic acid moiety or the saccharide moiety. J. Am. Chem. Soc., 117, at 8983. Shinkai's group showed that those disadvantages could be overcome by modifying the boronic acid binding site to create an electron rich center around the boronic acid moiety by, e.g., adding a tertiary amine, which formed an intramolecular five membered ring with the boronic acid moiety. Id. They then showed that the 9,10-bis-(aminomethyl)-anthracene skeleton provided a glucose selective sensor by producing a perfect glucose-selective cleft within the molecule. Id. These efforts resulted in the so called “Shinkai dyes” (FIG. 1A).
Building on the work of Shinkai's group, Norrild's group sought to create dyes that were both specific for the glucose molecule relative to other saccharides, and capable of binding glucose at neutral pH. Eggert et al, J. Org. Chem., 64, 3846-3852, 1999. To bind glucose at neutral pH, the boronic acid must have a pKa≦7. Devising such a boronic acid sensor dye proved to be a major problem in that most of the dyes proposed up to that point had pKa in the 8 to 10 range.
Norrild and co-workers chose 3-pyridineboronic acid for its especially low pKa, and produced a bis-boronic acid comprising two 3-pyridineboronic acid moieties grafted onto an anthracene moiety. The resulting “Norrild” dyes (FIG. 1B) possessed the requisite low pKa values and water solubility with a structurally optimized design for selective glucose binding. These dyes were reported to possess a selective fluorescence response to glucose compared to fructose and galactose.
Both the Shinkai and Norrild type dyes are solution-based systems. The solution-based system affords great freedom for the intramolecular changes that signal saccharide binding. However, certain applications require a solid-phase dye. Immobilization will likely reduce molecular freedom and alter the electrochemistry. This will in turn affect the fluorescence of the molecule, and might diminish its utility. That is, immobilization can reduce the fluorescence intensity or reduce the fluorescent response in the physiological testing region.
Saccharide sensors within the art generally have one or more shortcomings. For example, known sensors are capable of producing a discernible signal in response to saccharide binding, but do not provide the desired specificity for glucose relative to other saccharides.
Additionally, the known saccharide sensors lack the desired quantitative response. That is, as the concentration of saccharide increases, fluorescence increases only modestly. Preferably, the slope of relative fluorescence intensity as a function of saccharide concentration is high, and remains high throughout the range of saccharide concentration that is of physiological significance. Sensors exhibiting high slope of intensity, as a function of concentration will produce a signal that is more reliable and more readily discernible thereby substantially reducing costs of production.
Further, the use of fluorescence as a detection or diagnostic device benefits from a significant Stokes' shift. The Stokes' shift derives from the empirical law that the emission wavelength of a fluorescent material is longer than that of the radiation used to excite fluorescence. If the Stokes' shift is small, the excitation and emission peaks are in close proximity, and it is necessary to resort to sophisticated optics and filtering devices to distinguish the two. As the Stokes' shift increases, the wavelength of the excitation peak moves further away from emission peak, and the reliance on filters to distinguish the two is reduced. This again, facilitates a more economical device.
Attempts to improve the glucose specificity of boronic acid based saccharide sensors, and/or improve the fluorescence properties of the sensor, and/or immobilize those traditionally solution-based systems on polymeric substrates have met with only limited success. E.g., Arimori, S., et al, Chem. Commun., 2001, 1836-1837.
One of the principle objectives in devising a saccharide dye is to create a dye that demonstrates a steep slope for the curve of relative intensity versus saccharide concentration. Shinkai and Norrild have shown that it is possible to create saccharide dyes that, in a solution-based system, have that steep slope. Subsequent work, however, has shown that the covalent linkage of those dyes to a solid substrate can result in a substantial reduction in that slope. As yet, there is nothing within the art disclosing saccharide dyes covalently attached to solid-phase that do not suffer a substantial loss or elimination of that slope.
Some workers have attempted to extend the work of Shinkai and Norrild to achieving solid-phase construction, but fail to produce a system that can be used in direct blood contact, or in ex-vivo approaches, in which blood is circulated outside the body. WO 01/74968 A2 and WO 01/20334 A1. That work focuses on the use of an allegedly solid-phase saccharide dye of the Shinkai type to be inserted in a patient under the skin to measure saccharide in interstitial fluid spaces. See also U.S. Pat. Nos. 6,002,954, and 6,011,984.
There is a need within the art for saccharide dyes that have high glucose specificity. Ideally, such high specificity glucose sensors will demonstrate improved fluorescence properties. There is also a need in the art for dyes that retain such specificity and fluorescence properties when immobilized by covalent attachment to a solid substrate.